Lincomycin compounds

ABSTRACT

Novel useful analogs of the well known antibiotics lincomycin and clindamycin. These analogs are prepared by condensing a cyclic acid with a sugar amine.

DESCRIPTION CROSS REFERENCE TO RELATED APPLICATION

This is a continuation-in-part of my pending application Ser. No. 096,652, filed on Nov. 23, 1979, now abandoned, and is a division of application Ser. No. 148,056, filed May 19, 1980, now U.S. Pat. No. 4,278,789.

BACKGROUND OF THE INVENTION

The characteristics and preparation of the antibiotic lincomycin are disclosed in U.S. Pat. No. 3,086,912. Clindamycin is disclosed in U.S. Pat. No. 3,496,163. These antibiotics have been extensively used as medicines in humans and animals. A number of patents world-wide have issued concerning these antibiotics and a variety of derivatives thereof.

Lincomycin has the following structural formula ##STR1##

Clindamycin has the following structural formula ##STR2##

BRIEF SUMMARY OF THE INVENTION

This application relates to novel and useful compounds of the formula: ##STR3## wherein R₁, which can be singly or multiply substituted in any position of the pyridine ring not already substituted by R₂, is selected from the group consisting of hydrogen, alkyl and substituted alkyl wherein the alkyl portion is from 1 to 8 carbon atoms, inclusive, and isomeric forms thereof, cycloalkyl and substituted cycloalkyl, substituted oxygen, substituted nitrogen, halogen, phenyl, and substituted phenyl, --(CH₂)_(n) --OH, --(CH₂)_(n) --NR₄ R₅, and isomeric forms thereof, wherein n is an integer of from 1 to 8, inclusive, R₄ and R₅ are H or alkyl of from 1 to 8 carbon atoms, inclusive, and isomeric forms thereof, wherein R₂, which can be singly substituted in any position of the pyridine ring not already substituted by R₁, is ##STR4## and X is the amino function of a compound selected from the group consisting of 7(R)-hydroxy-methyl 1-thio-α-lincosaminide, 7(S)-hydroxy-methyl 1-thio-α-lincosaminide, 7(S)-halo-methyl 1-thio-α-lincosaminide, 7(R)-halo-methyl 1-thio-α-lincosaminide, 7(S)-methoxy-methyl 1-thio-α-lincosaminide, 7-deoxy-7(S)-(methylthio)-methyl 1-thio-α-lincosaminide, 7-deoxy-7(S)-(2-hydroxyethylthio)methyl 1-thio-α-lincosaminide, and 7-deoxy-7(S)-(3-hydroxypropylthio)-methyl 1-thio-α-lincosaminide; and the pharmaceutically acceptable acid addition salts thereof, and to novel compounds of the formula: ##STR5## wherein R₁ can be singly or multiply substituted in the ring on the same or different carbons, and R₂, which can be in the 2, 3, 4, 5, 6, 7, 8 or 9 positions of the ring, are as defined above; wherein R₃ is selected from the group consisting of H, CH₃, C₂ H₅, and --CH₂ --CH₂ --OH; wherein n is an integer of from 1 to 4, inclusive; and the pharmaceutically acceptable acid addition salts thereof.

Compounds of particular importance are of the formula: ##STR6## wherein R₁ is in the 4 position and is alkyl of from 1 to 8 carbon atoms, inclusive, and isomeric forms thereof; wherein R₃ is as defined above; wherein R₂ is in the 2 or 3 position and is otherwise as defined above; wherein n is 1; and the pharmaceutically acceptable acid addition salts thereof.

Important precursor compounds of the above have the formula ##STR7## wherein R₁ and R₂ are as defined immediately above.

The synthesis of the novel analogs described herein can be shown in exemplary form as follows: ##STR8## The wavy lines denote either the D-cis or L-cis isomer.

An alternate procedure which may be used to synthesize the novel analogs described herein can be shown in exemplary form as follows: ##STR9## The wavy line denotes either the D-cis, L-cis, D-trans or L-trans structures.

The L-cis structure ##STR10## has been shown to be 5 to 10 times more active than clindamycin against S. aureus and S. hemolyticus in laboratory mice.

An isomer of V may be isolated from the above reaction and is presumed to be the D-cis compound, VA. The D-cis structure is not as potent an antibacterial agent as the L-cis compound. ##STR11##

Also within the scope of the subject invention are compounds of the formulae ##STR12## wherein A, B, D and E are selected from the group consisting of nitrogen, oxygen, sulfur and CR₁ R₁ ; R₁ and R₂ are as defined previously and can be attached to any ring carbon or nitrogen atom; R₁ can be multiply attached to any ring carbon atom, and the pharmaceutically acceptable acid addition salts thereof.

Further, this application relates to novel 2-phosphates and 2-palmitates wherein the substitution is attached to the oxygen atom at the 2 position of the sugar ring of the above compounds.

DETAILED DESCRIPTION OF THE INVENTION

Upon reacting an amino acid of the formula ##STR13## wherein R₁, which can be singly or multiply substituted in any position of the pyridine ring not already substituted by ##STR14## is selected from the group consisting of hydrogen, alkyl and substituted alkyl wherein the alkyl portion is from 1 to 8 carbon atoms, inclusive, and isomeric forms thereof, cycloalkyl and substituted cycloalkyl, substituted oxygen, substituted nitrogen, halogen, phenyl and substituted phenyl; --(CH₂)_(n) --OH, --(CH₂)_(n) --NR₄ R₅, and isomeric forms thereof, wherein n is an integer of from 1 to 8, inclusive, R₄ and R₅ are H or alkyl of from 1 to 8 carbon atoms, inclusive, and isomeric forms thereof, wherein ##STR15## which can be singly substituted in any position of the pyridine ring not already substituted by R₁, with a sugar amine compound selected from the group consisting of 7(R)-hydroxy-methyl 1-thio-α-lincosaminide, 7(S)-hydroxy-methyl 1-thio-α-lincosaminide, 7(S)-halomethyl 1-thio-α-lincosaminide, 7(R)-halo-methyl 1-thio-α-lincosaminide, 7(S)-methoxy-methyl 1-thio-α-lincosaminide, 7-deoxy-7(S)-(methylthio)-methyl 1-thio-α-lincosaminide, 7-deoxy-7(S)-(2-hydroxyethylthio)-methyl 1-thio-α-lincosaminide, and 7-deoxy-7(S)-(3-hydroxypropylthio)-methyl 1-thio-α-lincosaminide; there are obtained novel and useful compounds of formula I.

Upon reacting an amino acid of the formula ##STR16## wherein R₁ and the position of substitution of ##STR17## are as defined above; wherein R₃ is selected from the group consisting of H, CH₃, C₂ H₅, and --CH₂ --CH₂ --OH; wherein n is an integer of from 1 to 4, inclusive, with a sugar amine compound, as defined above, there are obtained novel and useful compounds of formula II.

Upon reacting an acid of the formulae ##STR18## wherein A, B, D and E are selected from the group consisting of nitrogen, oxygen, sulfur and CR₁ R₁ ; R₁ is as defined previously and can be attached to any ring carbon or nitrogen atom; R₁ can be multiply attached to any ring carbon atom; ##STR19## can be attached to any ring carbon or nitrogen atom, with a sugar amine compound selected from the group as defined above, there are obtained novel and useful compounds of formulae VI and VII.

MTL is methyl 1-thio-α-lincosaminide of the formula ##STR20##

epi-MTL is methyl 7(S)-7-deoxy-7-hydroxy-1-thio-α-lincosaminide of the formula ##STR21##

7-Cl-MTL is methyl 7(S)-7-deoxy-7-chloro-1-thio-α-lincosaminide of the formula ##STR22##

epi-7-Cl-MTL is methyl 7(R)-7-deoxy-7-chloro-1-thio-α-lincosaminide of the formula ##STR23##

7(S)-methoxy-methyl 1-thio-α-lincosaminide can be shown as follows (See U.S. Pat. No. 3,702,322, Example 1, Part B-1): ##STR24##

With reference to the following formula, 7-deoxy-7(S)-(methylthio)-methyl 1-thio-α-lincosaminide exists when R is CH₃ ; 7-deoxy-7(S)-(2-hydroxyethylthio)-methyl 1-thio-α-lincosaminide exists when R is --CH₂ --CH₂ --OH; and, 7-deoxy-7(S)-(3-hydroxypropylthio)-methyl 1-thio-α-lincosaminide exists when R is --CH₂ --CH₂ --CH₂ --OH (See U.S. Pat. No. 3,915,954, Examples 1, 10 and 31): ##STR25##

The hydroxy and halo groups at the 7 position of the above formulas can be shown as follows ##STR26## wherein Y is selected from the group consisting of 7(R)-hydroxy, 7(S)-hydroxy, 7(S)-halo, and 7(R)-halo.

When a pyridine acyl group is used, the resulting analog can be reduced to give a mixture of the corresponding saturated compounds, one of which is the L-cis isomer. Other compounds which may be present include the L-trans, D-cis, and D-trans isomers. Generally, for any of the compounds described herein, the reduced form is more antibacterially-active than the unsaturated precursor. The use of a piperidine acyl group gives analogs existing as D-cis, L-cis, D-trans, and L-trans isomers. Again, the L-cis isomer has been found to be more anti-bacterially active.

The general method used herein to prepare the novel analogs is the well known process wherein an appropriate acid is coupled with an appropriate sugar amine. ("Mixed Carboxylic Acid Anhydride Procedure," Chemistry of The Amino Acids, Vol. 2, p. 970, John Wiley and Sons, Inc. 1961.) When the acid is unsaturated, the resulting unsaturated analog can be catalytically reduced under standard conditions to prepare the saturated analog. For example, the reduction can be conducted using the following conditions:

H₂ at 5 to 50 psi

Catalyst--platinium oxide (PtO₂)

Solvent--H₂ O or H₂ O+MeOH, or H₂ O+EtOH

HCl--10% excess

Time--24 to 48 hours

As used herein, alkyl of 1 to 8 carbon atoms, inclusive, and isomeric forms thereof, includes methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, and branched chain isomers thereof.

Substituted alkyl means the above alkyl compounds in which one or more of the hydrogen atoms has been replaced by a halogen, i.e., Cl, Br, F, and I, oxygen, hydroxyl, amine (primary), amine (secondary-alkyl substituted by alkyl as above), amine (tertiary-alkyl substituted by alkyl as above), sulfur, --SH, and phenyl. Exemplary compounds are 1-fluoroethyl, 1-chloroethyl, 2-fluoroethyl, 2-chloroethyl, 1-bromopropyl, 2-iodopropyl, 1-chlorobutyl, 4-fluorobutyl, and 4-chlorobutyl.

Cycloalkyl means cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.

Substituted cycloalkyl means a cycloalkyl substituted as above for substituted alkyl. Exemplary compounds are 2-cyclopropylethyl, 3-cyclobutylpropyl, 4-cyclopentylbutyl, and 4-cyclohexylbutyl.

Aromatic means phenyl and substituted phenyl wherein one or more of the hydrogen atoms has been replaced by a halogen, as above, hydroxyl, amine (primary, secondary, and tertiary with the latter two alkyl substituted as above), --SH, and phenyl. Exemplary compounds are p-bromophenyl, m-iodophenyl, o-chlorophenyl, p-ethylpheryl, m-propylphenyl, o-methylphenyl, and p-octylphenyl.

As detailed infra, the compounds of the invention can be phosphorylated to give the 2-phosphate, and acylated to give the 2-palmitate which are both antibacterially-active in vivo.

Substituted oxygen means oxygen substituted by an alkyl of from 1 to 8 carbons, inclusive, aryl, and substituted aryl.

Substituted nitrogen means nitrogen substituted by an acyl of from 2 to 18 carbons, a monoalkyl of 1-8 carbons, inclusive, and a dialkyl, wherein the alkyl is from 1 to 8 carbons, inclusive, including the isomeric forms for all acyl and alkyl groups.

Halo means chloro, bromo, iodo, or fluoro.

Exemplary sources for the amino acids used as starting materials herein are as follows:

1. Heterocyclic Compounds, Vol. 1, John Wiley and Sons, Inc., 1950. This source describes the preparation of halogen and alkyl substituted amino acids.

2. Chem. Abstracts:

81--105223A--alkyl and cycloalkyl

81--152243S--alkyl and halogen substituted

82--170746H--halogen substituted

85--46322Q--dihalo substituted

85--177258W--dihalo substituted

84--116928X--dihalo substituted

81--3737d--phenyl substituted

78--58201t--phenyl substituted

76--126800y--tetrahalo substituted

82--11036K--bromo substituted

83--27119W--bromo substituted

84--16613X--bromo substituted

78--123494G--bromo substituted

    ______________________________________                                         84 - 135488V -                                                                               ##STR27##                                                        81 - 151951J -                                                                               ##STR28##                                                        81 - 77809a -                                                                                ##STR29##                                                        84 - 30918G -                                                                                ##STR30##                                                        81 - 33139C -                                                                                ##STR31##                                                        79 - 19109V -                                                                                ##STR32##                                                        81 - 3737D -                                                                                 ##STR33##                                                        81 - 135964K -                                                                               ##STR34##                                                        85 - 177349B -                                                                               ##STR35##                                                        78 - 71865G -                                                                                ##STR36##                                                        81 - 135964K -                                                                               ##STR37##                                                        83 - 147397G -                                                                               ##STR38##                                                        82 - 11036K -                                                                                ##STR39##                                                        84 - 116928X -                                                                               ##STR40##                                                        81 - 33139C -                                                                                ##STR41##                                                        76 - 126800Y -                                                                               ##STR42##                                                        79 - 115449b -                                                                               ##STR43##                                                        67 - 63229K -                                                                                ##STR44##                                                        68 - 104926b                                                                                 ##STR45##                                                        69 - 59048Z                                                                                  ##STR46##                                                        71 - 124907M                                                                                 ##STR47##                                                                      ##STR48##                                                        68 - 59465N                                                                                  ##STR49##                                                        ______________________________________                                    

This compound can be hydrolyzed to the acid by means well known in the art, which acid can then be reduced, also by means well known in the art. ##STR50## This compound can be hydrolyzed to the acid by means well known in the art. The resulting acid then can be N-demethylated by the procedures disclosed in U.S. Pat. No. 3,583,972. ##STR51## This compound can be hydrolyzed to the acid by means well known in the art. ##STR52## This compound can be hydrolyzed to the acid by means well known in the art. Also, one or both of the N--CH₃ groups can be removed from the resulting acid by following the procedures disclosed in U.S. Pat. No. 3,583,972. ##STR53## This compound can be hydrolyzed to the acid by means well known in the art. The resulting acid can be converted to the following compound ##STR54## by methods disclosed in U.S. Pat. No. 3,583,972. ##STR55##

Compounds having free NH₂ or OH groups will have to have these groups protected before being condensed with the amino sugar. Protection of such groups is well known in the art. See Protective Groups in Organic Chemistry, J. F. W. McOmie, Plenum Publishing Co., Ltd., 1973.

3. Jour. Chem. Soc. 1969-2134--Various H-alkyl substituted pyridines ##STR56## R=alkyl, branched alkyl and cycloalkyl 4. Jour. Chem. Soc. 1969-934-- ##STR57##

The following examples are illustrative of the process and products of the invention, but are not to be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

EXAMPLE 1 4-Cis-ethyl-L-pipecolic acid amide of 7-Cl-MTL.HCl (U-57,930E-Compound V) PART I ##STR58##

A solution of 67 g (0.357 moles) of the amino acid HCl (C.A. 51, 1643a, 1957) and 71.5 g (0.714 moles) of triethylamine dissolved in 2.5 liters of acetonitrile is cooled to 10° C. and 47.6 g (0.354 moles) of isobutylchloroformate added in one portion. This mixture (Solution A) is stirred at 10° C. for 1 hour. Solution B is made up by dissolving 97.7 g (0.357 moles) of 7-Cl-MTL (J. Med. Chem., 12-780, 1969, B. J. Magerlein and F. Kagan) in a warm mixture of 1500 ml of acetone and 1500 ml of H₂ O. Solution B is cooled to 30° C. and added in one portion to Solution A. The reaction is stirred at 25° C. for 18 hours and the acetone and acetnoitrile removed under vacuum. The white, mushy residue is filtered and the crystalline material collected and dried to give 95 g of pure product. Workup of the filtrate (chromatography) gave another 10 g of product. The overall yield is 73%. Anal. Calcd. for C₁₇ H₂₅ ClN₂ O₅ S: C, 50.42; H, 6.22; N, 6.92; S, 7.92; Cl, 8.76. Found: C, 50.67; H, 6.40; N, 6.64; S, 7.90; Cl, 8.70.

α_(D) ^(CHCl).sbsp.3 : (C, 1.0)+293°.

PART II ##STR59##

A mixture of 4.05 g (0.01 mole) of starting material, 40 ml of water, 60 ml of methanol, 1.0 ml of 37% HCl and 8.0 g of PTO₂ catalyst were reduced on a Parr hydrogenator at 50 p.s.i. for 3 hours. Analysis of the reaction mixture by TLC on silica gel plates in a system composed of CHCl₃ :methanol (6:1) showed that all of the starting material was gone and that two more polar materials were present in a ratio of about 1:1. The reaction was filtered to remove the catalyst and the filtrate concentrated under vacuum to give a white crystalline mush. This was filtered and the filtrate saved. The white solid, which was the most polar of the two products observed upon TLC of the reduction mixture, was recrystallized from water to give the desired product, U-57,930E, m.p. 222°-224°, in a yield of from 25 to 35%. Anal. Calcd. for C₁₇ H₃₂ Cl₂ N₂ O₅ S: C, 45.63; H, 7.21; N, 6.26; S, 7.17; Cl, 15.85. Found: C, 45.77; H, 7.44; N, 6.39; S, 7.21; Cl, 16.17.

α_(D) ^(H).sbsp.2^(O) : (C, 1.0)+176°.

The absolute configuration and sterochemistry of V was established by X-ray crystallography.

U-57,930E, tested in comparison with clindamycin, has the following antimicrobial spectra:

                  TABLE I                                                          ______________________________________                                         The Minimal Inhibitory Concentration of U-57,930E                              and Clindamycin Vs. Aerobic Bacteria.                                                        MIC (μg/ml)                                                   Organism   UC       Clindamycin  U-57,930E                                     ______________________________________                                         Staphylococcus                                                                            6685     >25          >25                                           aureus     6686     .05          .20                                                      6687     .025         .20                                                      6688     >25          >25                                                      6689     .05          .78                                                      6690     .025         .20                                                      6691     .10          .20                                                      6692     >25          >25                                                      6693     .05          .78                                                      6694     >25          >25                                                      6695     .10          .39                                                      6696     .10          .39                                                      6675     .05          .39                                                       76      .05          .10                                                       746     ≦.05  .05                                                       571     .20          .78                                                       570     .20          .39                                           Staphylococcus                                                                             719     .10          .20                                           epidermidis                                                                               3389     .10          .20                                           Streptococcus                                                                  faecalis    694     25           6.25                                          Streptococcus                                                                  pyogenes    152     ≦.012 ≦.012                                  Streptococcus                                                                  viridans    153     ≦.012 .05                                                       871     ≦.012 ≦.012                                  Diplococcus                                                                    pneumoniae I                                                                               41      ≦.012 ≦.012                                  Diplococcus                                                                    pneumoniae II                                                                             3213     ≦.012 ≦.012                                  Escherichia coli                                                                           45      50           >50                                           Proteus vulgaris                                                                           93      >50          >50                                           Klebsiella                                                                     pneumoniae  58      6.25         >50                                           Salmonella                                                                     schottmuelleri                                                                             126     >50          >50                                           Pseudomonas                                                                    aeruginosa  95      >50          >50                                           ______________________________________                                    

The procedure for the above test is as follows:

The Minimal Inhibitory Concentration (MIC's) of both compounds Vs. aerobic bacteria is determined using a standard microplate broth-dilution method. Brain Heart Infusion (BHI-Difco) broth medium is used, and the plates are incubated at 37 C. for 20 hrs.

S. aureus UC 6685-6696 are clinical isolates which are resistant to one or more commercial antibiotics.

"UC" is a registered trademark of The Upjohn Company Culture Collection. These cultures can be obtained from The Upjohn Company in Kalamazoo, Mich., upon request.

                  TABLE II                                                         ______________________________________                                         The Minimal Inhibitory Concentration of Clindamycin and                        U-57930E vs. Gram-Positive and Gram-Negative Anaerobic                         Bacteria                                                                                       MIC(μg/ml)                                                  Organism     UC       Clindamycin  U-57930E                                    ______________________________________                                         Bacteroides  6513     0.06         0.12                                        fragilis     6428     0.06         0.25                                                     6864     3.9          2.0                                                      6862     7.8          15.6                                        Bacteroides                                                                    thetaiotaomicron                                                                            6512     2.0          0.5                                         Bacteroides                                                                    distasonis   6518     0.12         ≦0.03                                Bacteroides                                                                    melaninogenicus                                                                             6326     0.06         0.06                                        Clostridium                                                                    perfringens   247     0.06         0.12                                                     6509     0.06         0.12                                        Clostridium                                                                    novyi B      6329     0.06         0.12                                        Clostridium                                                                    tertium      6508     7.8          7.8                                         Clostridium                                                                    cadaveris    6510     ≦0.03 0.06                                        Clostridium                                                                    sordellii    6505     2.0          0.5                                         Clostridium                                                                    tentani      6521     ≦0.03 ≦0.03                                Clostridium                                                                    botulinum A  6506     0.25         ≦0.03                                Clostridium                                                                    bifermentans 6507     0.50         0.06                                        Clostridium                                                                    difficile    6834     7.8          3.9                                                      6857     250          125                                                      6858     3.9          3.9                                                      6860     500          500                                                      6861     3.9          2.0                                         Propionibacterium                                                              acnes        6564     0.06         0.12                                                     6575     <0.03        0.06                                        Eubacterium                                                                    limosum      6515     2.0          2.0                                         Eubacterium                                                                    lentum       6522     0.50         1.0                                         Actinomyces                                                                    naeslundii   5920     0.25         0.25                                        Fusobacterium                                                                  nucleatum    6516     0.12         0.12                                                     6324     0.06         0.06                                        Fusobacterium                                                                  varium       6052     15.6         3.9                                         Fusobacterium                                                                  necrophorum  6568     0.06         0.06                                        Peptococcus                                                                    asaccharolyticus                                                                            6214     0.05         0.25                                        Peptococcus                                                                    magnus       6258     0.06         0.06                                        Peptococcus                                                                    aerogenes    6319     ≦0.03 0.06                                        Peptostreptococcus                                                             anaerobius   6321     0.12         0.12                                        ______________________________________                                    

The procedure for the above test is as follows: Serial two-fold dilutions of drug are prepared in 1.0 ml volumes of Schaedler Broth, and 9.0 ml of molten (47° C.) Wilkens-Chalgren Agar Medium, infra, is added to the antibiotic-supplemented broth. After mixing with the antibiotic, the agar is poured into 100 mm×20 mm petri dishes. The dishes are allowed to stand on the bench overnight prior to inoculation.

Cultures are streaked on Wilkens-Chalgren Agar, and grown for 48 hours at 37° C. in a BBL Anaerobe Jar. Growth from the plate is harvested, and a cell suspension is made in Schaedler broth to equal the turbidity of a 0.5 McFarland Standard (10⁸ cells/ml). The suspension is pipetted into the wells of a Steers replicator, and approx. 1-2 μl is delivered to the surface of the agar plates. After allowing a few minutes for the inoculum to dry, the plates are placed in a BBL Anaerobe Jar (atmosphere of 85% N, 10% H, 5% CO₂) and incubated at 37° C. for 72 hours.

The Minimal Inhibitory Concentration (MIC) is read as the least amount of drug that inhibits growth. A very faint film of growth, or <3 colonies is considered negative.

Wilkins-Chalgren Agar Medium

Dispense the following ingredients and dissolve in 1000 ml distilled water. The pH should be 7.0-7.2.

Trypticase: 10 g

Gelysate: 10 g

Yeast Extract: 5 g

Glucose: 1 g

NaCl: 5 g

L-Arginine-Free Base: 1 g

Pyruvic Acid-Sodium Salt: 1 g

Agar: 15 g

Add Heme and +Vitamin K₁ solutions to yield final concentrations of 5 μg/ml Hemin and 0.5 g/ml K₁

Autoclave at 121° C. for 15 minutes aerobically. Heme Stock-0.5 g Hemin+10 ml 1 N NaOH+990 ml H₂

Autoclave at 121° C. for 12 minutes.

Add 10 ml stock per liter of medium. +Vitamin K Stock-0.05 Vit. K₁ solution+20 ml 95% ethanol

Filter sterilize

Add 0.2 ml stock per liter of medium

The I.P. LD₅₀ of U-57,930E in the mouse was found to be 592 mg/kg. This value is the resulting mean of two separate and identical LD₅₀ determinations. This value is approximately 2 times the LD₅₀ for clindamycin HCl. The LD₅₀ value should be interpreted as indicating that the acute I.P. toxicity of U-57,930E is approximately one-half that of clindamycin HCl.

    ______________________________________                                         In vivo : Mouse Protection Test                                                Organism & Route                                                                            Clindamycin U-57930A    Ratio                                     ______________________________________                                         S. aureus                                                                      Subcut.       5.7(4.2-7.8)*                                                                             <<5         --                                        Oral         12.3(8.8-17.3)                                                                             1-5         ca. 10                                    S. hemolyticus                                                                 Subcut.       2.3(1.6-3.3)                                                                              .25         ca. 10                                    Subcut.       3.3(2.6-4.2)                                                                              .25(0.2-0.33)                                                                              13                                        Oral         12.3(10.2-14.8)                                                                            2.9(2.0-4.1  4.2                                      K. pneumoniae                                                                                 >320      >320        --                                        ______________________________________                                          *CD.sub.50 's as mg/kg                                                   

The procedure for the above test is as follows:

Mouse-protection Tests

Groups of 10 standard laboratory mice (CF-1 Mice) weighing 18-20 gm were infected with approximately 100 LD₅₀ 's of standardized bacterial cell suspensions which had been maintained frozen at -170° C. Immediately before use, the suspensions were thawed quickly and properly diluted. Infection was via the intraperitoneal route.

Treatment of the infected groups was begun immediately and continued once per day for 4 days (first 24 hr period=1). Groups of untreated infected mice served as virulence controls for the culture.

Seven days after the treatment regimen was begun the surviving animals were sacrificed and the median protective dose of the antibiotic calculated on the basis of mortality rates in the treatment groups. The median protective dose and its 95% confidence interval were calculated according to the method of Spearmen & Karber as programmed on a 360 digital computer.

Also isolated from Example I, Part II, is compound V A. This material is obtained as follows:

The filtrate which was saved from Part II was concentrated to dryness under vacuum, the residue converted to its free base and chromatographed over silica gel using CHCl₃ :methanol (6:1) as the eluting solvent. In this manner the least polar material mentioned in Part II was obtained. It was converted to its HCl salt and recrystallized from acetone and water. This isomer is tentatively being assigned structure V A. ##STR60##

Epimerization of the carbonyl function attached to the piperidine ring of V and V A may be accomplished by methods well known to those skilled in the art. The trans isomers V B and V C produced by these epimerizations may be isolated by conventional procedures such as crystallization or chromatography. ##STR61##

Alternatively, V and V A may be hydrolized to give the amino acids V D and V E which may then be epimerized by methods well known to those skilled in the art to V F and V G, respectively. The amino acids V F and V G may be coupled with any of the lincosaminides described earlier. ##STR62##

The D-cis isomer (V A) of U-57,930E has an antibacterial spectrum when tested on BHl broth as described previously in Table 1.

    ______________________________________                                         Organism       UC No.   MIC (μg/ml)                                         ______________________________________                                         S. aureus       76        250                                                                 570       1000                                                                 746        125                                                  S. fecalis     694      >1000                                                  S. pyogenes    152        62.5                                                 D. pneumoniae   41        62.5                                                 E. coli         45      >1000                                                  K. pneumoniae   58      >1000                                                  S. schottmuelleri                                                                             126      >1000                                                  Ps. aeruginosa  95      >1000                                                  ______________________________________                                    

EXAMPLE 2 Other Analogs of 7-Cl-MTL

By following the procedures of Example 1, but substituting the amino acid with the following amino acids there are prepared the corresponding novel antibacterially-active analogs as their free bases or acid addition salts. The latter can be prepared by methods well-known to those skilled in the art.

    ______________________________________                                         Amino Acid        Analog                                                       ______________________________________                                          ##STR63##        U-45,863                                                      ##STR64##        U-46,138                                                      ##STR65##        U-46,137                                                      ##STR66##        U-46,337 (Fast isomer on TLC- MeOH:CHCl 95:5. Run on                           silica gel plates)                                            ##STR67##        U-46,465 (Slow isomer on TLC)                                 ##STR68##        U-46,699 (Slow isomer on TLC, prepared from U-46,465)         ##STR69##        U-45,656                                                      ##STR70##        U-45,652                                                      ##STR71##        U-46,701                                                      ##STR72##        U-60,481                                                      ##STR73##        U-44,469                                                      ##STR74##        U-45,657                                                     ______________________________________                                    

EXAMPLE 3 Analogs of MTL

By following the procedures of Example 1, but substituting the amino acid with the following amino acids and substituting MTL (J. Am. Chem. Soc., 89-2448, 1967 W. Schroeder, B. Bannister and H. Hoeksema) for 7-Cl-MTL, there are prepared the corresponding novel antibacterially-active analogs:

    ______________________________________                                         Amino Acid      Analogs                                                        ______________________________________                                          ##STR75##      U-46,136                                                        ##STR76##      U-45,653                                                        ##STR77##      U-60,493 (fast isomer TLC)                                      ##STR78##      U-60,492 (slow isomer TLC)                                     ______________________________________                                    

EXAMPLE 4 Analogs of epi-MTL

By following the procedures of Example 1, but substituting the amino acid with the following amino acids, and substituting epi-MTL (J. Chem. Soc. Perkin I 1974, p. 360-B, Bannister) for 7-Cl-MTL there are prepared the corresponding novel antibacterially-active analogs:

    ______________________________________                                         Amino acid           Analog                                                    ______________________________________                                          ##STR79##           U-46,135                                                   ##STR80##           U-45,659                                                   ##STR81##           Cpd. A                                                     ##STR82##           Cpd. B.                                                   ______________________________________                                    

EXAMPLE 5 Analogs of epi-7-Cl-MTL

By following the procedures of Example 1, but substituting the amino acid with the following amino acids, and substituting epi-7-Cl-MTL for 7-Cl-MTL, there are prepared the corresponding novel antibacterially-active analogs:

    ______________________________________                                         Amino Acid           Anaolg                                                    ______________________________________                                          ##STR83##           Cpd. C                                                     ##STR84##           Cpd. D                                                     ##STR85##           Cpd. E                                                     ##STR86##           Cpd. F                                                    ______________________________________                                    

Epi-7-Cl-MTL can be prepared by the procedure used to prepare 7-Cl-MTL with the exception that the starting material is epi-MTL instead of MTL.

Chemical and physical characterization of most of the compounds of Examples 2-5 are as follows:

    ______________________________________                                                 C    H      N      S    Cl   αD                                                                             Mp.                                 ______________________________________                                         U-45,863                                                                               1     47.80  5.62 7.44 8.51 9.41 CHCl.sub.3   --                               2     47.76  5.54 7.35 8.76 9.34 +286°  96-100°          U-46,138                                                                               1     47.80  5.62 7.44 8.51 9.41  --   --                                      2     47.30  5.74 6.91 8.51 9.44                                       U-46,137                                                                               1     47.80  5.62 7.44 8.51 9.41 EtOH                                          2     47.58  5.74 7.56 8.53 9.49 +216° 189-190°          U-46,136                                                                               1     50.26  6.19 7.82 8.95      MeOH                                          2     49.16  5.86 8.01 9.15      +194° 199-201°          U-46,135                                                                               1     50.26  6.19 7.82 8.95      MeOH                                          2     50.50  6.19 7.98 9.27      +269°  97-100°          U-46,337A                                                                              1     42.96  6.73 6.68 7.65 16.91                                                                               EtOH                                          2     42.73  6.86 6.52 7.73 16.68                                                                               +206° 220-30°           U-46,465E                                                                              1     47.05  7.11 7.32 8.38 9.26 EtOH                                          2     46.63  7.37 7.12 8.47 9.33 +231° 180-3°            U-46,699E                                                                              1     44.34  6.98 6.47 7.40 16.36                                                                               H.sub.2 O                                     2     44.79  7.24 6.25 7.36 16.42                                                                               +172°  229-234°         U-45,656                                                                               1     50.42  6.22 6.92 7.92 8.76 CHCl.sub.3                                    2     50.85  6.39 6.72 7.54 8.93 +250°   --                     U-45,652                                                                               1     50.42  6.22 6.92 7.92 8.76 CHCl.sub.3                                    2     51.03  6.40 6.65 7.56 8.02 +273°   --                     U-45,653                                                                               1     52.83  6.78 7.25 8.30 --   MeOH                                          2     53.83  7.08 7.39 8.14 --   +203°   --                     U-45,659                                                                               1     52.83  6.78 7.25 8.30 --   CHCl.sub.3                                    2     52.77  6.70 7.34 8.55 --   +295°   --                     U-46,701A                                                                              1     46.85  7.43 6.07 6.95 15.37                                              2     --     --   --   --   --    --   --                              U-44,469E                                                                              1     44.06  6.96 6.05 6.92 15.30                                              2     44.83  6.68 6.07 6.72 15.49                                                                                --   --                              U-45,657                                                                               1     43.80  4.90 6.81 7.80 17.24                                                                               MeOH                                          2     43.52  4.93 6.82 7.82 17.41                                                                               °181° 105-130.degre                                              e.                                    ______________________________________                                          1  CALCD.                                                                      2  FOUND                                                                 

EXAMPLE 6 Fusaric Acid Amide of 7-Chloro-MTL ##STR87##

By following the procedure of Example 1, but substituting the amino acid with fusaric acid, there is obtained U-55,581.

Anal. Calcd. for C₁₉ H₂₉ ClN₂ O₅ S: C, 52.70; H, 6.75; N, 6.47; S, 7.41; Cl, 8.19. Found: C, 52.15; H, 6.65; N, 6.36; S, 7.21; Cl, 7.94.

EXAMPLE 7 4-Cis-n-Butyl-L-Pipecolic Acid Amide of 7-Cl-MTL or U-50,970E ##STR88##

A mixture of 4.0 g (0.0093 mole) of starting material, 40 ml of water, 40 ml of methanol, 2 ml of 37% HCl, and 8.0 g. PtO₂ catalyst were reduced on a Parr hydrogenator at 50 psi for 18 hours. The reaction was filtered to remove the catalyst and the filtrate concentrated under vacuum to give an amber oil. The oil was dissolved in 20 ml. of a 2:1 solution of CHCl₃ and methanol and enough triethylamine added to neutralize the HCl present. This solution was then chromatographed over silica gel using a solvent system composed of CHCl₃ :methanol (2:1). Two main product fractions are obtained. The fractions containing the faster moving material were pooled and evaporated under vacuum to give a white solid, fraction A. The fractions containing the slower moving material were pooled and evaporated under vacuum to give a white solid, fraction B. Fraction B was dissolved in a small amount of H₂ O and enough 37% HCl added to make the pH 2. Crystallization occurred. The solid was collected and recrystallized from H₂ O to give white crystals of the desired product, U-60,970E, m.p. 224°-226° in a yield of 25-35%.

Anal. Calcd. for C₁₇ H₃₂ Cl₂ N₂ O₅ S: C, 47.99; H, 7.63; N, 5.89; S, 6.75; Cl, 14.92. Found: C, 47.97; H, 7.42, N, 6.23; S. 6.90; Cl, 14.87.

α_(D) ^(MeOH) : +178° (C, 1.0).

CMR analysis supports the proposed structure.

The Minimal Inhibitory Concentration (MIC) in μg/ml of U-60,970E against various bacteria is as follows:

    ______________________________________                                         Organism       UC#         MIC                                                 ______________________________________                                         S. aureus      76          0.125                                                              570         0.25                                                               746         0.062                                               S. faecalis    694         0.25                                                S. pyogenes    152         0.008                                               D. pneumoniae  41          0.016                                               E. coli        45          31.2                                                K. pneumoniae  58          7.8                                                 S. schottmuelleri                                                                             126         31.2                                                Ps. aeruginosa 95          >125                                                ______________________________________                                    

The test procedure is as disclosed in Example 1.

U-60,970E was also tested in vivo in standard laboratory mice which were experimentally infected with bacteria. The test was conducted in comparison with U-57,930E. The following results show that U-60,970E is significantly more active in vivo against D. pneumoniae I and III than U-57,930E. Against S. aureus and S. hemolyticus U-60,970E demonstrates essentially the same activity as U-57,930E.

    __________________________________________________________________________                    Median Portective Dose (CD.sub.50 in mg/kg)                                    U-57930E . Hcl    U-60970E . Hcl                                Organism  Strain                                                                              Subcutaneous                                                                           Oral      Subcutaneous                                                                           Oral                                  __________________________________________________________________________     S. hemolyticus                                                                           C203 0.38 (0.28-0.51)                                                                        3.79 (2.74-5.29)                                                                        0.35    2.18 (1.5-3.16)                       S. aureus UC 76                                                                               1.07 (0.72-1.59)                                                                        8.12 (5.78-11.41)                                                                       1.32 (1.05-1.66)                                                                       8.12 (6.75-9.77)                      D. pneumoniae I                                                                          Felton I                                                                            1.54 (1.12-2.11)                                                                       15.39 (10.57-22.4)                                                                       0.67 (0.52-0.86)                                                                       4.42 (3.15-6.21)                      D. pneumoniae III                                                                        MPI III                                                                             2.68 (1.88-3.81)                                                                       14.36 (9.68-21.31)                                                                       0.44 (0.36-0.54)                                                                       3.13 (2.26-4.32)                                     2.5  (1.69-3.69)                                                                       16.49 (10.51-25.88)                                                                      0.29 (0.21-0.41)                                                                       4.12 12.93-5.81)                      __________________________________________________________________________

EXAMPLE 8 4-Cis-n-Butyl-D-Pipecolic Acid Amide of 7-Cl-MTL or U-61,734E ##STR89## Fraction A from the preceding experiment was converted to its HCl salt in the same manner as described for fraction B. A 25-35% yield of product was obtained whose CMR spectrum was essentially identical to that obtained from fraction B. EXAMPLE 9 Preparation of a Compound in Which the Aminoacid Portion Contains a Heteroatom IN a 5-Membered Ring ##STR90##

The aminoacid ester (see C.A. 69-67282M) may be hydrolyzed to the free acid by methods well known to those skilled in the art (acid or basic hydrolysis may be used). It may be obtained in the form of the .HCl salt or the zwitterion. The coupling of the aminoacid .HCl with 7-Cl-MIL is accomplished in the same manner as described in Example 1, except that 67.7 g. (0.357 moles) of the aminoacid is used. After workup, as described in Example 1, the crude product may be purified via chromatography over silica gel and the product fractions combined and converted to the HCl salt.

EXAMPLE 10 Preparation of a Compound in Which the Aminoacid Portion Contains a Heteroatom in a 6-Membered Ring ##STR91##

The aminoacid ester (see C.A. 68-59465N) may be hydrolyzed to the free acid by methods well known to those skilled in the art (acid or basic hydrolysis may be used). It may be obtained in the form of the HCl salt or the zwitterion. The coupling of the aminoacid .HCl with 7-Cl-MTL is accomplished in the same manner as described in Example 1 except that 103.6 g. (0.357 moles) of the aminoacid is used. After workup, as described in Example 1, the crude product may be purified via chromatography over silica gel and the product fractions combined and converted to the HCl salt.

EXAMPLE 11 2-Phosphate Analogs

The 2-phosphate analog of the compounds prepared in Examples 1-10 can be prepared by procedures well-known to those skilled in the art. By obvious appropriate modification, the procedure disclosed in U.S. Pat. No. 3,487,068 may be used. Basically, any procedure would first involve the protection of vulnerable groups by methods well-known to those skilled in the art which would then be removed upon completion of the phosphorylation.

EXAMPLE 12 2-Palmitate Analogs

The 2-palmitate analog of the compounds prepared in Examples 1-10 can be prepared by procedures well-known to those skilled in the art. By obvious appropriate modification, the procedure disclosed in U.S. Pat. No. 3,580,904 may be used. Basically, any procedure would first involve the protection of vulnerable groups by methods well-known to those skilled in the art which would then be removed upon completion of the acylation with palmitoyl chloride.

The Minimal Inhibitory Concentration (MIC's) of a representative number of the compounds prepared in Examples 2-8 follows. The test procedure is as given supra.

    __________________________________________________________________________     MIC in mcg/ml                                                                  U-Number and Structure                                                         __________________________________________________________________________                 45,656  45,652                                                                               45,653                                                                               45,659                                                                               46,701  44,469                                                                               45,657                                  ##STR92##                                                                              ##STR93##                                                                            ##STR94##                                                                            ##STR95##                                                                            ##STR96##                                                                              ##STR97##                                                                            ##STR98##                 __________________________________________________________________________     S. aureus UC 76                                                                            62.5    1000  >1000 >1000 62.5    250   125                        S. aureus UC 570                                                                           62.5    >1000 >1000 >1000 125     250   250                        S. aureus UC 746                                                                           62.5    1000  >1000 1000  625     >1000 250                        S. hemolyticus UC 152                                                                      3.9     500   >1000 500    --     31.3  7.8                        St. faecalis UC 694                                                                        1000    >1000 >1000 >1000 500     1000  >1000                      S. lutea UC 130                 1000                                           E. coli UC 45                                                                              >1000   >1000 >1000 >1000 >1000   >1000 >1000                      P. vulgaris UC 93                                                                          >1000   >1000 >1000 >1000 >1000   >1000 >1000                      K. pneumoniae UC 58                                                                        1000    500   >1000 1000  >1000   >1000 >1000                      S. schottmuelleri UC 126                                                                   1000    >1000 >1000 >1000 >1000   >1000 >1000                      Ps. aeruginosa UC 95                                                                       >1000   >1000 >1000 1000  >1000   >1000 >1000                      D. pneumoniae UC 41                                                                         --     500   >1000 500    --     31.3   --                        __________________________________________________________________________      ##STR99##                                                                      ##STR100##                                                                     ##STR101##                                                                     R = C.sub.2 H.sub.5- -                                                   

                45,863                                                                               46,138                                                                               46,137                                                                             46,136                                                                               46,135                                                                               46,337                                                                               46,465                                                                               46,699                                  ##STR102##                                                                           ##STR103##                                                                           ##STR104##                                                                         ##STR105##                                                                           ##STR106##                                                                           ##STR107##                                                                           ##STR108##                                                                           ##STR109##                __________________________________________________________________________     S. aureus UC 76                                                                            62.5        >1000                                                                              >1000 >1000 1000  3.9   3.9                        S. aureus UC 570        >1000                                                                              >1000 >1000 1000  31.2  7.8                        S. aureus UC 746        >1000                                                                              >1000 >1000 1000  3.9   7.8                        S. hemolyticus UC 152                                                                      2.0   Qual. 1000                                                                               1000  1000  250   <1.0  3.9                        St. faecalis UC 694                                                                        >1000 Assay >1000                                                                              >1000 >1000 >1000 3.9   15.6                                         Inactive                                                     E. coli UC 45                                                                              >1000 vs.   1000                                                                               >1000 >1000 >1000 >1000 1000                       P. vulgaris UC 93                                                                          >1000 S. lutea                                                                             >1000                                                                              >1000 >1000 >1000 1000  1000                       K. pneumoniae UC 58                                                                        >1000       1000                                                                               >1000 >1000 >1000 500   250                        S. schottmuelleri UC 126                                                                   >1000       1000                                                                               >1000 >1000 >1000 1000  >1000                      Ps. aeruginosa UC 95                                                                       >1000       1000                                                                               >1000 >1000 >1000 >1000 >1000                      D. pneumoniae UC 41                                                                        62.5        500 >1000 >1000 250   <1.0  <1.0                                                               Fast  Slow  N-methyl                                                           isomer                                                                               isomer                                                                               of                                                                 on TLC                                                                               on TLC                                                                               slow                                                                           isomer                     __________________________________________________________________________      ##STR110##                                                                     ##STR111##                                                                     ##STR112##                                                                    - -                                                                       

                                          U-60,493                                                                             U-60,492                                                                             U-60,481                                                                  ##STR113##                                                                           ##STR114##                  __________________________________________________________________________                               S. aureus UC 76                                                                            1000  1000  2.0                                                    S. aureus UC 570                                                                           1000  >1000 3.9                                                    S. aureus UC 746                                                                           250   250   2.0                                                    S. hemolyticus UC 152                                                                      7.8   15.6  >1.0                                                   St. faecalis UC 694                                                                        1000  >1000 31.2                                                   S. lutea UC 130                                                                E. coli UC 45                                                                              >1000 >1000 500                                                    P. vulgaris UC 93                                                                          >1000 >1000 1000                                                   K. pneumoniae UC 58                                                                        >1000 >1000 250                                                    S. Schottmuelleri UC 126                                                                   >1000 >1000 1000                                                   Ps. aeruginosa UC 95                                                                       >1000 >1000 >1000                                                  D. pneumoniae UC 41                                                                        15.6  31.2   --                                                                Fast  Slow  Ir &                                                               isomer                                                                               isomer                                                                               NMR OK                                                             on TLC                                                                               on TLC                             __________________________________________________________________________      ##STR115##                                                                     ##STR116##                                                                     R = C.sub.2 H.sub.5                                                      

Since the compounds of the subject invention are active against various Gram-positive and Gram-negative microbes, they can be used in various environments to inhibit such microbes. For example, they can be used as disinfectants to inhibit S. aureus on washed and stacked food utensils contaminated with this bacterium. They also can be used as disinfectants on various dental and medical equipment contaminated with S. aureus. Further, the compounds of the invention can be used as bacteriostatic rinses for laundered clothes, and for impregnating papers and fabrics; and, they are also useful for suppressing the growth of sensitive organisms in plate assays and other microbiological media.

The compounds of the subject invention exist in the protonated or non-protonated forms according to the pH of the environment. When the protonated form is intended, the compounds exist as pharmaceutically-acceptable acid-addition salts, and when the non-protonated form is intended, the compounds exist as the free base. The free bases can be converted to stable acid-addition salts by neutralizing the free base with the appropriate acid, about pH 7.0, and advantageously to about pH 2 to pH 6. Suitable acids for this purpose include hydrochloric, sulfuric, phosphoric, thiocyanic, fluosilicic, hexafluoroarsenic, hexafluorophosphoric, acetic, succinic, citric, lactic, maleic, fumaric, pamoic, cholic, palmitic, mucic, camphoric, glutaric, glycolic, phthalic, tartaric, lauric, stearic, salicyclic, 3-phenylsalicyclic, 5-phenylsalicyclic, 3-methylglutaric, orthosulfobenzoic, cyclohexanesulfamic, cyclopentanepropionic, 1,2-cyclohexanedicarboxylic, 4-cyclohexanecarboxylic, octadecenylsuccinic, octenylsuccinic, methanesulfonic, helianthic, Reinecke's, dimethyldithiocarbamic, hexadecylsulfamic, octadecylsulfamic, sorbic, monochloroacetic, undecylenic, 4'-hydroxyazobenzene-4-sulfonic, octadecylsulfuric, picric, benzoic, cinnamic, and like acids.

The acid-addition salts can be used for the same purposes as the free base or they can be employed to upgrade the same. For example, the free base can be converted to a water-insoluble salt, such as the picrate, which can be subjected to purification procedures, for example, solvent extractions and washings, chromatography, fractional liquid-liquid extractions, and crystallization, and then used to regenerate the free base form by treatment with alkali or to make a different salt by metathesis. Or the free base can be converted to a water-soluble, salt, such as the hydrochloride or sulfate, and the aqueous solution of the salt extracted with various water-immiscible solvents before regenerating the free base form by treatment of the thus-extracted acid solution, or converted to another salt by metathesis.

In addition to the antibacterial uses, disclosed above, the free bases can be used as buffers or as antacids. The thiocyanic acid addition salt when condensed with formaldehyde forms resinous materials useful as pickling inhibitors according to U.S. Pat. Nos. 2,425,320 and 2,606,155. The free bases also make good vehicles for toxic acids. For example, the fluosilicic acid addition salts are useful as mothproofing agents according to U.S. Pat. Nos. 1,915,334 and 2,075,359 and the hexafluoroarsenic acid and hexafluorophosphoric acid addition salts are useful as parasiticides according to U.S. Pat. Nos. 3,122,536 and 3,122,552.

The compounds of the subject invention are useful as antibacterial agents in suitable compositions. These compositions are preferably presented for administration to humans and animals in unit dosage forms, such as tablets, capsules, pills, powders, granules, sterile parenteral solutions or suspensions, and oral solutions or suspensions, and oil-water emulsions containing suitable quantities of the active compound in the form of the free base, or its pharmacologically acceptable salts.

For oral administration, either solid or fluid unit dosage forms can be prepared. For preparing solid compositions such as tablets, the principal active ingredient is mixed with conventional ingredients such as talc, magnesium stearate, dicalcium phosphate, magnesium aluminum silicate, calcium sulfate, starch, lactose, acacia, methylcellulose, and functionally similar materials as pharmaceutical diluents or carriers. The tablets can be laminated or otherwise compounded to provide a dosage form affording the advantage of prolonged or delayed action or predetermined successive action of the enclosed medication. For example, the tablet can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permits the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids or mixture of polymeric acids with such materials as shellac, cetyl alcohol, cellulose acetate phthalate, styrene maleic acid copolymer and the like. Alternatively, the two component system can be utilized for preparing tablets containing two or more incompatible active ingredients. Wafers are prepared in the same manner as tablets, differing only in shape and the inclusion of sucrose or other sweetener and flavor. In their simplest embodiment, capsules, like tablets, are prepared by mixing the compound of the formulation with an inert pharmaceutical diluent and filling and mixture into a hard gelatin capsule of appropriate size. In another embodiment, capsules are prepared by filling hard gelatin capsules with polymeric acid coated beads containing the active compound. Soft gelatin capsules are prepared by machine encapsulation of a slurry of the active compound with an acceptable vegetable oil, light liquid petrolatum or other inert oil.

Fluid unit dosage forms for oral administration such as syrups, elixirs, and suspensions can be prepared. The water-soluble forms of the active compound can be dissolved in an aqueous vehicle together with sugar, aromatic flavoring agents and preservatives to form a syrup. An elixir is prepared by using a hydro-alcoholic (ethanol) vehicle with suitable sweeteners such as sucrose together with an aromatic flavoring agent. Suspensions can be prepared of the insoluble forms with a syrup vehicle with the aid of a suspending agent such as acacia, tragacanth, methylcellulose and the like.

Topical ointments can be prepared by dispersing the active compound in a suitable ointment base such as petrolatum, lanolin, polyethylene glycols, mixtures thereof, and the like. Advantageously, the compound is finely divided by means of a colloid mill utilizing light liquid petrolatum as a levigating agent prior to dispersing in the ointment base. Topical creams and lotions are prepared by dispersing the compound in the oil phase prior to the emulsification of the oil phase in water.

For parenteral administration, fluid unit dosage forms are prepared utilizing the active compound and a sterile vehicle, water being preferred. The active compound, depending on the form and concentration used, can be either suspended or dissolved in the vehicle. In preparing solutions, a water-soluble form of the active compound can be dissolved in water for injection and filter sterilized before filling into a suitable vial or ampul and sealing. Advantageously adjuvants such as a local anesthetic, preservation and buffering agents can be dissolved in the vehicle. To enhance the stability, the composition can be frozen after filling into the vial and the water removed under vacuum. The dry lyophilized powder is then sealed in the vial and an accompanying vial of water for injection is supplied to reconstitute the powder prior to use. Parenteral suspensions are prepared in substantially the same manner except that the active compound is suspended in the vehicle instead of being dissolved and sterilization cannot be accomplished by filtration. The active compound can be sterilized by exposure to ethylene oxide before suspending the sterile vehicle. Advantageously, a surfactant or wetting agent is included in the composition to facilitate uniform distribution of the active compound.

The term unit dosage form as used in the specification and claims refers to physically discrete units suitable as unitary dosages for human subjects and animals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical diluent, carrier or vehicle. The specifications for the novel unit dosage forms of this invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active material for therapeutic use in humans and animals, as disclosed in detail in this specification, these being features of the present invention. Examples of suitable unit dosage forms in accord with this invention are tablets, capsules, pills, troches, suppositories, powder packets, granules, wafers, cachets, teaspoonfuls, tablespoonfuls, dropperfuls, ampuls, vials, segregated multiples of any of the foregoing, and other forms as herein described.

In addition to the administration of the active compound as the principal active ingredient of compositions for the treatment of the conditions described herein, the said compound can be included with other types of compounds to obtain advantageous combinations of properties. Such combinations include the active compound with antibiotics such as spectinomycin, chloramphenicol, novobiocin, dihydronovobiocin, tetracyclines (e.g., tetracycline, oxytetracycline and chlortetracycline) penicillins, erythromycin, kanamycin, streptomycin, neomycin, polymyxin, bacitracin, nystatin, filipin, fumagillin and endomycin to broaden the bacterial spectrum of the composition and for synergistic action against particular bacteria; steroids having anti-inflammatory activity such as hydrocortisone, prednisolone, 6α-methylprednisolone, 6α-fluoroprednisolone and the like; analgesics such as aspirin, sodium salicylate (acetylsalicyclic acid)-anhydride, N-acetyl-p-aminophenyl and salicylamide; antihistamines, such as chlorpheninamine maleate, diphenylhydramine, promethazine, pyrathiazine, and the like; sulfas, such as sulfadiazine, sulfamethazine, sulfamerazine sulfacetamide, sulfadimethyloxazole, sulfamethizole, and the like; antifungals, such as undecylenic acid, sodium propionate, salicylanilide, sodium caprylate, and hexetidine; and the vitamins.

The dosage of the active compound for treatment depends on route of administration; the age, weight, and condition of the patient; and the particular disease to be treated. A dosage schedule of from about 15 to 500 mg., 1 to 4 times daily (every six hours), embraces the effective range for the treatment of most conditions for which the compositions are effective. For children, the dosage is calculated on the basis of 15 to 30 mg./kg./day to be administered every six hours.

The active compound is compounded with a suitable pharmaceutical carrier in unit dosage form for convenient and effective administration. In the preferred embodiments of this invention, the dosage units contain the compound in: 15, 30, 50, 125, 250 and 500 mg. amounts for systemic treatment; in 0.25, 0.5, 1, 2 and 5% amounts for topical or localized treatment; and 5 to 65% w/v for parenteral treatment. The dosage of compositions containing the active compound and one or more other active ingredients is to be determined with reference to the usual dosage of each such ingredient.

The following examples are illustrative of the best mode contemplated by the inventor for carrying out his invention and are not to be construed as limiting.

The examples use U-57,930E or U-60,970E as the active compound, but it should be understood that this is only exemplary of the other active compounds of the subject invention. As distinguished from the previous examples, the following are Composition Examples.

COMPOSITION EXAMPLE 1 Capsules

One thousand two-piece hard gelatin capsules for oral use, each containing 250 mg. of U-57,930E or U-60,970E are prepared from the following types and amounts of materials:

    ______________________________________                                                           Gm.                                                          ______________________________________                                         U-57,930E or U-60,970E                                                                             250                                                        Corn starch         100                                                        Talc                 75                                                        Magnesium stearate   25                                                        ______________________________________                                    

The materials are thoroughly mixed and then encapsulated in the usual manner.

The foregoing capsules are useful for the systemic treatment of infection in adult humans by the oral administration of 1 capsule every 4 hours.

Using the procedure above, capsules are similarly prepared containing U-57,930E or U-60,970E in 15, 30, 50, 125 and 500 mg. amounts by substituting 15, 30, 50, 125, and 500 gm of U-57,930E or U-60,970E for the 250 gm used above.

COMPOSITION EXAMPLE 2 Capsules

One thousand two-piece hard gelatin capsules for oral use, each containing 200 mg. of U-57,930E or U-60,970E and 250 mg. of tetracycline hydrochloride, are prepared from the following types and amounts of ingredients:

    ______________________________________                                                            Gm.                                                         ______________________________________                                         U-57,930E or U-60,970E                                                                              200                                                       Tetracycline hydrochloride                                                                          250                                                       Talc                  75                                                       Magnesium stearate    25                                                       ______________________________________                                    

The ingredients are thoroughly mixed and then encapsulated in the usual manner.

The foregoing capsules are useful for the systemic treatment of infection in adult humans by the oral administration of 1 capsule every 6 hours.

Using the procedure above, capsules are similarly prepared containing U-57,930E or U-60,970E and each of the following antibiotics in place of tetracycline by substituting 250 gm. of such other antibiotic for tetracycline: chloramphenicol, oxytetracycline, chlortetracycline, fumagillin, erythromycin, streptomycin, dihydronovobiocin and novobiocin. When a penicillin, such as potassium penicillin G, is to be used in place of tetracycline, 250,000 units per capsule is employed.

Such combination products are useful for the systemic treatment of mixed infections in adult humans by the oral administration of 1 capsule every 6 hours.

COMPOSITION EXAMPLE 3 Tablets

One thousand tablets for oral use, each containing 500 mg. of U-57,930E or U-60,970E are prepared from the following types and amounts of materials:

    ______________________________________                                                           Gm.                                                          ______________________________________                                         U-57,930E or U-60,970E                                                                             500                                                        Lactose             125                                                        Corn starch         65                                                         Magnesium stearate  25                                                         Light liquid petrolatum                                                                            3                                                          ______________________________________                                    

The ingredients are thoroughly mixed and slugged. The slugs are broken down by forcing through a number sixteen screen. The resulting granules are then compressed into tablets, each tablet containing 500 mg. of U-57,930E or U-60,970E.

The foregoing tablets are useful for systemic treatment of infections in adult humans by oral administration of 1 tablet every 4 hours.

Using the above procedure, except for reducing the amount of U-57,930E or U-60,970E to 250 gm., tablets containing 250 mg. of U-57,930E or U-60,970E are prepared.

COMPOSITION EXAMPLE 4 Tablets

One thousand oral tablets, each containing 250 mg. of U-57,930E or U-60,970E, and a total 250 mg. (83.3 mg. each) of sulfadiazine, sulfamerazine, and sulfamethazine, are prepared from the following types and amounts of materials:

    ______________________________________                                                           Gm.                                                          ______________________________________                                         U-57,930E or U-60,970E                                                                             250                                                        Sulfadiazine        83.3                                                       Sulfamerazine       83.3                                                       Sulfamethazine      83.3                                                       Lactose             50                                                         Corn starch         50                                                         Calcium stearate    25                                                         Light liquid petrolatum                                                                            5                                                          ______________________________________                                    

The ingredients are thoroughly mixed and slugged. The slubs are broken down by forcing through a number sixteen screen. The resulting granules are then compressed into tablets, each containing 250 mg. of U-57,930E or U-60,970E and a total of 250 mg. (83.3 mg. each) of sulfadiazine, sulfamerazine, and sulfamethazine.

The foregoing tablets are useful for systemic treatment of infections by the oral administration of 4 tablets first and then 1 every six hours.

For the treatment of urinary infections, the triple sulfas in the above formulation is advantageously replaced by 250 gm. of sulfamethylthiadiazole or 250 gm. of sulfacetamide.

COMPOSITION EXAMPLE 5 Oral Syrup

One thousand cc. of an aqueous suspension for oral use, containing in each 5 cc. dose, one-half gram of total sulfas and 250 mg. of U-57,930E or U-60,970E is prepared from the following types and amounts of ingredients:

    ______________________________________                                                                Gm.                                                     ______________________________________                                         U-57,930E or U-60,970E   50                                                    Sulfadiazine             33.3                                                  Sulfamerazine            33.3                                                  Sulfamethazine           33.3                                                  Citric acid              2                                                     Benzoic acid             1                                                     Sucrose                  700                                                   Tragacanth               5                                                     Lemon oil 2 cc.                                                                Deionized water, q.s. 1,000 cc.                                                ______________________________________                                    

The citric acid, benzoic acid, sucrose, tragacanth, and lemon oil dispersed in sufficient water to make 850 cc. of solution. The U-57,930E or U-60,970E and finely powdered sulfas are stirred into the syrup until uniformly distributed. Sufficient water is added to make 1,000 cc.

The composition so prepared is useful in the systemic treatment of pneumonia in adult humans at a dose of 1 teaspoonful 4 times a day.

COMPOSITION EXAMPLE 6 Parenteral Solution

A sterile aqueous solution for intramuscular use, containing in 1 cc. 200 l mg. of U-57,930E or U-60,970E is prepared from the following types and amounts of materials:

    ______________________________________                                                                Gm.                                                     ______________________________________                                         U-57,930E or U-60,970E   200                                                   Lidocaine hydrochloride  4                                                     Methylparaben            2.5                                                   Water for injection, q.s. 1,000 cc.                                            ______________________________________                                    

The ingredients are dissolved in water and the solution sterilized by filtration. The sterile solution is filled into vials and the vials sealed.

COMPOSITION EXAMPLE 7 Parenteral Preparation

A sterile aqueous solution for intramuscular use, containing in 1 cc. 200 mg. of U-57,930E and U-60,970E and 400 mg. of spectinomycin sulfate, is prepared from the following types and amounts of ingredients:

    ______________________________________                                                             Gm.                                                        ______________________________________                                         U-57,930E or U-60,970E                                                                               200                                                      Spectinomycin sulfate 400                                                      Lactose                50                                                      Water for injection, q.s. 1,000 cc.                                            ______________________________________                                    

The U-57,930E or U-60,970E, spectinomycin sulfate and lactose are dissolved in the water and the solution sterilized by filtration. The sterile solution, in the amount of 2 cc., is aseptically filled into sterile vials and frozen. The water is removed under high vacuum and the vials containing the lyophilized powder are sealed. Just prior to use, sufficient sterile water for injection to make 2 cc. of solution is added to the vial.

COMPOSITION EXAMPLE 8 Topical Ointment

One thousand gm. of 0.25% ointment is prepared from the following types and amounts of ingredients:

    ______________________________________                                                             Gm.                                                        ______________________________________                                         U-57,930E or U-60,970E                                                                               2.5                                                      Zinc oxide            50                                                       Calamine              50                                                       Liquid petrolatum (heavy)                                                                            250                                                      Wool fat              200                                                      White petrolatum, q.s. 1,000 gm.                                               ______________________________________                                    

The white petrolatum and wool fat are melted and 100 gm. of liquid petrolatum added thereto. The U-57,930E or U-60,970E, zinc oxide and calamine are added to the remaining liquid petrolatum and the mixture milled until the powders are finely divided and uniformly dispersed. The powder mixture is stirred into the white petrolatum mixture and stirring continued until the ointment congeals.

The foregoing ointment is usefully applied topically to the skin of mammals for the treatment of infection.

The foregoing composition can be prepared by omitting the zinc oxide and calamine.

Following the procedure above, ointments are similarly prepared containing U-57,930E or U-60,970E in 0.5, 1, 2, and 5% amounts by substituting 5, 10, 20 and 50 gm. of U-57,930E or U-60,970E or the 2.5 gm. used above.

COMPOSITION EXAMPLE 9 Cream

One thousand gm. of a vaginal cream are prepared from the following types and amounts of ingredients:

    ______________________________________                                                                Gm.                                                     ______________________________________                                         U-57,930E or U-60,970E   50                                                    Tegacid Regular.sup.1    150                                                   Spermaceti               100                                                   Propylene glycol         50                                                    Polysorbate 80           5                                                     Methylparaben            1                                                     Deionized water, q.s. 1,000 gm.                                                ______________________________________                                          .sup.1 Self-emulsifying glyceryl monostearate from Goldschmidt Chemical        Corporation, New York, N.Y.                                              

The Tegacid and spermaceti are melted together at a temperature of 70°-80° C. The methylparaben is dissolved in about 500 gm. of water and the propylene glycol Polysorbate 80, and U-57,930E or U-60,970E are added in turn, maintaining a temperature of 75°-80° C. The methylparaben mixture is added slowly to the Tegacid and spermaceti melt, with constant stirring. The addition is continued for at least 30 minutes with continued stirring until the temperature has dropped to 40°-45° C. The pH of the final cream is adjusted to 3.5 by incorporating 2.5 l gm. of citric acid and 0.2 g. of dibasic sodium phosphate dissolved in about 50 gm. of water. Finally, sufficient water is added to bring the final weight to 1,000 gm. and the preparation stirred to maintain homogeneity until cooled and congealed.

The foregoing composition is useful for the treatment of vaginal infections in humans.

COMPOSITION EXAMPLE 10 Ointment, Ophthalmic

One thousand gm. of an ophthalmic ointment containing 0.5% U-57,930E or U-60,970E are prepared from the following types and amounts of ingredients:

    ______________________________________                                                                 Gm.                                                    ______________________________________                                         U-57,930E or U-60,970E    5                                                    Bacitracin                12.2                                                 Polymyxin B sulfate (10,000                                                    units/mg.)                1                                                    Light liquid petrolatum   250                                                  Wool fat                  200                                                  White petrolatum, q.s. 1,000 gm.                                               ______________________________________                                    

The solid ingredients are finely divided by means of an air micronizer and added to the light liquid petrolatum. The mixture is passed through a colloid mill to uniformly distribute the micronized particles. The wool fat and white petrolatum are melted together, strained, and the temperature adjusted to 45°-50° C. The liquid petrolatum slurry is added and the ointment stirred until congealed. Suitably the ointment is packaged in one dram opthalmic tubes.

The foregoing ointment is usefully applied to the eye for treatment of localized infection in humans and other animals.

Advantageously the foregoing composition can contain 5 gm. (0.5%) of methylprednisolone for the treatment of inflammation, and, alternatively, the bacitracin and polymyxin B sulfate can be omitted.

COMPOSITION EXAMPLE II Eye-Ear Drops

One thousand cc. of a sterile aqueous solution for eye or ear use containing 10 mg. of U-57,930E or U-60,970E and 5 mg. of methylprednisolone in each cc. is prepared from the following types and amounts of ingredients:

    ______________________________________                                                                 Gm.                                                    ______________________________________                                         U-57,930E or U-60,970E    10                                                   Methylprednisolone phosphate sodium                                                                      5                                                    Sodium citrate            4.5                                                  Sodium bisulfite          1                                                    Polyethylene glycol 4000  120                                                  Myristyl-γ-picolinium chloride                                                                     0.2                                                  Polyvinylpyrrolidone      1                                                    Deionized water, q.s. ad 1000 cc.                                              ______________________________________                                    

The ingredients are dissolved in the water and the resulting solution is sterilized by filtration. The solution is aseptically filled into sterile dropper containers.

The composition so prepared is useful in the topical treatment of inflammation and infection of the eye and ear as well as other sensitive tissues of the animal body.

COMPOSITION EXAMPLE 12 Troches

Ten thousand troches are prepared from the following types and amounts of ingredients:

    ______________________________________                                                                 Gm.                                                    ______________________________________                                         U-57,930E or U-60,970E    100                                                  Neomycin sulfate          50                                                   Polymyxin B sulfate (10,000                                                    units/mg.)                1                                                    Ethyl aminobenzoate       50                                                   Calcium stearate          150                                                  Powdered sucrose, q.s. 5,000 gm.                                               ______________________________________                                    

The powdered materials are mixed thoroughly and then compressed into half gram troches following the usual techniques for the preparation of compressed tablets.

The troches are held in the mouth and allowed to dissolve slowly to provide treatment for the mouth and throat of humans.

COMPOSITION EXAMPLE 13 Suppository, Rectal

One thousand suppositories, each weighing 2.5 gm. and containing 100 mg. of U-57,930E or U-60,970E are prepared from the following types and amounts of ingredients:

    ______________________________________                                                                Gm.                                                     ______________________________________                                         U-57,930E or U-60,970E   100                                                   Polymyxin B sulfate (10,000                                                    units/mg.)               1.25                                                  Methylprednisolone       1                                                     Ethyl aminobenzoate      75                                                    Zinc oxide               62.5                                                  Propylene glycol         162.5                                                 Polyethylene                                                                   glycol 4,000 q.s. 2,500 gm.                                                    ______________________________________                                    

The U-57,930E or U-60,970E, polymyxin B sulfate methylprednisolone, ethyl aminobenzoate, and zinc oxide are added to the propylene glycol and the mixture milled until the powders are finely divided and uniformly dispersed. The polyethylene glycol 4000 is melted and the propylene glycol dispersion added slowly with stirring. The suspension is poured into unchilled molds at 40° C.

The composition is allowed to cool and solidify and then removed from the mold and each suppository foil wrapped.

The foregoing suppositories are inserted rectally for local treatment of inflammation and infection.

Alternatively, the foregoing composition can be prepared omitting the steroid.

COMPOSITION EXAMPLE 14 Mastitis Ointment

One thousand gm. of an ointment for the treatment of mastitis in dairy cattle is prepared from the following types and amounts of ingredients

    ______________________________________                                                                 Gm.                                                    ______________________________________                                         U-57,930E or U-60,970E    25                                                   Methylprednisolone acetate                                                                               0.5                                                  Light liquid petrolatum   300                                                  Chlorobutanol, anhydrous  5                                                    Polysorbate 80            5                                                    2% Aluminum monostearate-                                                      peanut oil gel            400                                                  White petrolatum, q.s. 1000 gm.                                                ______________________________________                                    

The U-57,930E or U-60,970E and methylprednisolone acetate are milled with the light liquid petrolatum until finely divided and uniformly dispersed. The chlorobutanol, polysorbate 80, peanut oil gel and white petrolatum are heated to 120° F. to form a melt and the liquid petrolatum dispersion stirred in. With continued stirring, the dispersion is allowed to cool (and congeal) to room temperature and is filled into disposable mastitis syringes in 10 gm. doses.

COMPOSITION EXAMPLE 15 Animal Feed

One thousand gm. of a feed mix is prepared from the following types and amounts of ingredients:

    ______________________________________                                                               Gm.                                                      ______________________________________                                         U-57,930E or U-60,970E  10                                                     Soybean meal            400                                                    Fish meal               400                                                    Wheat germ oil          50                                                     Sorghum molasses        140                                                    ______________________________________                                    

The ingredients are mixed together and pressed into pellets. The composition can be fed to laboratory animals, i.e., rats, mice, guinea pigs, and hamsters for prophylaxis during shipping.

For other animals such as poultry, e.g., chickens, ducks, turkeys, and geese, the composition can be added to the animal's regular feed in an amount calculated to give the desired dose of U-57,930E or U-60,970E.

COMPOSITION EXAMPLE 16

Following the procedure of each of the preceding Composition Examples 1-15, inclusive, each antibacterially-active compound of the subject invention is substituted in an equivalent amount for the U-57,930E or U-60,970E shown in the example to provide therapeutic properties.

Similarly, each of the above free base compounds can be used in the form of a pharmaceutically (or pharmacologically) acceptable acid addition salt, e.g., hydrochloride, sulfate, nitrate, phosphate, citrate, lactate, acetate, tartrate and succinate.

Further, the 2-phosphate and/or 2-palmitate of each of the above antibacterially-active invention compounds can be substituted as the active ingredient to provide compositions having therapeutic properties. 

I claim:
 1. A compound of the formula ##STR117## wherein A, B, D and E are selected from the group consisting of nitrogen, oxygen, sulfur and CR₁ R₁ ; wherein R₁ is selected from the group consisting of hydrogen, alkyl and substituted alkyl wherein the alkyl portion is from 1 to 8 carbon atoms, inclusive, and isomeric forms thereof, cycloalkyl and substituted cycloalkyl, substituted oxygen, substituted nitrogen, halogen, phenyl, and substituted phenyl, --(CH₂)_(n) --OH, --(CH₂)_(n) --NR₄ R₅, and isomeric forms thereof, wherein n is an integer of from 1 to 8, inclusive, R₄ and R₅ are H or alkyl of from 1 to 8 carbon atoms, inclusive, and isomeric forms thereof; wherein R₂ is ##STR118## and X is the amino function of a compound selected from the group 7(R)-hyroxy-methyl 1-thio-α-lincosaminide, 7(S)-hydroxy-methyl 1-thio-α-lincosaminide, 7(S)-halo-methyl 1-thio-α-lincosaminide, 7(R)-halo-methyl 1-thio-α-lincosaminide, 7(S)-methoxy-methyl 1-thio-α-lincosaminide, 7-deoxy-7(S)-(methylthio)-methyl 1-thio-α-lincosaminide, 7-deoxy-7(S)-(2-hydroxyethylthio)-methyl 1-thio-α-lincosaminide and 7-deoxy-7(S)-(3-hydroxypropylthio)-methyl 1-thio-α-lincosaminide; R₁ and R₂ can be attached to any ring carbon or nitrogen atom; R₁ can be multiply attached to any ring carbon atom, and the pharmaceutically acceptable acid addition salts thereof.
 2. A compound, according to claim 1 of the formula ##STR119## and the pharmaceutically acceptable acid-addition salts thereof.
 3. The 2-phosphate of the compounds defined in claim
 1. 